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Should I dilute cDNA for real-time PCR? - (Feb/06/2006 )

Hi all ...

I'm in the process of performing "real time RT-PCR", I'm just a beginner to this field. What I did so far was I took 4 diffrent samples from the same tissue, same species. After total RNA extraction, I used 6ug to make first-strand cDNA's and stored it at -20 C.

Q: Am I supposed to dilute the cDNA concentration to (e.g 100ng/ul) before I run the real time RT-PCR if my aim was to compare the gene expression of these 4 diffrent samples? Whats the reasoning behind this, if its true?

I did use 2ul of 100ng/ul and ran a RT-PCR to test the specificity of this sample and the band size and intensity was good after 30 cycles.

Any suggestions would be highly appreciated.

Best regards

-Zezo-

Real time pcr is very sensative. Less is best in this case.

I typically use 1ug of total RNA in the cDNA reaction (20ul volume) then dilute 1:10 with depc water.

I will use 5 ul of that in a 25 ul real time pcr reaction.

-pBluescript-

Here is what you want to do:

Dilute the product from your reverse transcription reaction at least five or ten-fold to remove the PCR inhibitory effects of RT. If you need to use this sample many times and a ten-fold dilution is insufficient, you can go up to a hundred fold dilution without influencing your results deleteriously.

Pipette one to two microliters from your diluted cDNA sample into each 25 ul reaction volume. I personally use two microliters because I don't always have a one microliter pipettor handy, but one microliter is more than enough.

You also need to define a standard for quantifying between samples. Beta-actin is a good one, as is GAPDH (Beta actin is better, though). Order primers for this standard and create a vector containing the cloned standard for easy quantification.

-Matt

-MisticMatt-

i'm talking about this in another thread, i've been doing real-time pcr for 2 years and didn't know this! no one [including the guy from biorad who trained me] told me you had to dilute the cDAN. with undiluted cDNA most of the primers i have designed have worked, and the primer efficiencies are bang on 95-100%.... weird... first time i diluted my cDNA i got a primer efficiency of 120%!!!!

-flashboy-